Adenylated dinucleotides(ApxA)s have been termed alarmones because they are released from blood platelets following stress and modulate gene or enzyme activities. In support of this hypothesis our laboratory has demonstratedAp4A (the most abundant and best characterized Ap4A) is a signaling device that triggers the synthesis of protein(s)required to deliver L-Arg to bovine aortic endothelial cell (BAEC) nitric oxide synthase to generate nitric oxide. Our laboratory has also demonstrated that Ap4A is internalized by BAEC via endocytosis and this internalized Ap4A is not degraded. These data support the hypothesis that the internalizedAp4A may have a physiological role in vascular biology. We have also demonstrated that Ap4A acts as a partial agonist for ATP. These data suggest that Ap4Amay be a modulator of the physiological responses elicited by ATP. Thus, Ap4A truly fits the definition of an alarmone alerting the cells to the onset of metabolic stress and subsequently regulating gene expression and/or enzyme function. In this proposal we will use suppression subtractive hybridization to isolateAp4A differentially expressed cDNA clones and compare their sequences to Gen-Bank database. This comparision will give us information on what metabolic processes that Ap4A up regulates. We will also characterize the mechanism by which Ap4A is internalized and also determine the intracellular localization of the internalizedAp4A. We will accomplish this by determining whether inhibitors of endoctosis inhibit Ap4A internalization and whetherAp4A can be found associated with caveolae or clathrin-coated vesicles. In addition we will determine the time dependency of Ap4A uptake into various subcellular fractions or into the extracellular spaces. We will also determine whether Ap4A induces the release of calcium from calcium release channels on the endoplasmic reticulum. The information obtained in this proposal will give us insight on how to develop future proposals, biochemical and molecular experiments to determine the mechanism(s)by which Ap4A acts as an alarmone.